Molecularly-imprinted material made by template-directed synthesis

ABSTRACT

A method of making a molecularly imprinted porous structure makes use of a surfactant analog of the molecule to be imprinted that has the imprint molecule portion serving as the surfactant headgroup. The surfactant analog is allowed to self-assemble in a mixture to create at least one supramolecular structure having exposed imprint groups. The imprinted porous structure is formed by adding reactive monomers to the mixture and allowing the monomers to polymerize, with the supramolecular structure serving as a template. The resulting solid structure has a shape that is complementary to the shape of the supramolecular structure and has cavities that are the mirror image of the imprint group. Similarly, molecularly imprinted particles may be made by using the surfactant to create a water-in-oil microemulsion wherein the imprint groups are exposed to the water phase. When reactive monomers are allowed to polymerize in the water phase to form particles, the surface of the particles have cavities that are the mirror image of the imprint group.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates generally to molecularly-imprinted materialand in particular to molecularly-imprinted material made bytemplate-directed synthesis.

[0003] 2. Description of the Related Art

[0004] Enzymes are commonly exploited for practical uses, including ascatalysts in synthetic processes, as detection reagents in chemical andbiological sensors, and as catalysts in decontamination of environmentalpollutants and other toxic agents. Their usefulness is largely due totheir exquisite functional selectivity and regio- and stereospecificity.However, the usefulness of enzymes for practical purposes is limited bytheir intolerance to harsh conditions, particularly to conditionsinvolving nonaqueous environments, temperature extremes, or the presenceof materials that are toxic to the enzyme. In addition, enzymes may havea short shelf-life under ambient conditions and may require refrigeratedstorage to remain active.

[0005] Similarly, antibodies are useful for practical purposes such asfor detecting or separating specific materials in complex mixtures. Aswith enzymes, the usefulness of antibodies is due to their functionalselectivity and regio- and stereospecificity. Also, as with enzymes,their usefulness is limited by their intolerance for harsh conditions.

[0006] As a result of the difficulties in exploiting enzymes andantibodies on a large scale and in harsh environments, efforts have beenmade to develop enzyme and antibody mimics, that is, materials that canfunction as enzymes or antibodies, but which have a more durablecomposition. Specifically, efforts have been made to utilize theprinciples of molecular recognition to create artificial enzyme activesites or antibody binding sites through molecular imprinting of enzymetransition state analogs or antibody antigens in polymers and inorganicmatrices. For example, molecularly printed materials are described inthe following patents and publications incorporated herein by reference:U.S. Pat. No. 5,110,833 to Mosbach; U.S. Pat. No. 5,310,648 to Arnold etal; U.S. Pat. No. 5,372,719 to Afeyan et al; U.S. Pat. No. 5,453,199 toAfeyan et al; U.S. Pat. No. 5,461,175 to Fischer et al; U.S. Pat. No.5,587,273 to Yan et al; U.S. Pat. No. 5,630,978 to Domb; U.S. Pat. No.5,641,539 to Afeyan et al; U.S. Pat. No. 5,728,296 to Hjertén et al;U.S. Pat. No. 5,750,065 to Kilbane II; U.S. Pat. No. 5,756,717 toPaliwal et al; U.S. Pat. No. 5,786,428 to Arnold et al; U.S. Pat. No.5,814,223 to Hjertén et al; U.S. Pat. No. 5,821,311 to Mosbach et al;U.S. Pat. No. 5,858,296 to Domb; U.S. Pat. No. 5,872,198 to Mosbach etal.; Mosbach, K. et al, “The Emerging Technique of Molecular imprintingand Its Future Impact on Biotechnogy”, Biotechnology, vol 14, February1996, pp 163-170; G. Wulff, “Molecular Imprinting in Cross-LinkedMaterials with the Aid of Molecular Templates—A Way towards ArtificialAntibodies”Angew. Chem. Intl. Ed. Engl., 34, 1812-1832 (1995); P.Hollinger, et al., “Mimicking Nature and Beyond” Trends in Biochemistry,13(1), 7 9 (1995); Haupt, K., Mosbach, K. Trends Biotech, 16, 468-475(1997); Davis et al, “Rational Catalyst Design via ImprintedNanostructured Materials” Chem. Mater. 8 (1996) pp 1820-1839. and Wulff.G. et al, “Enzyme models Based on Molecularly Imprinted Polymers withStrong Esterase Activity” Angew. Chem. Int. Ed. Engl., 36 1962 (1997).

[0007] During a typical imprinting process, a molecule to be imprintedis combined with a mixture of functionalized and non-functionalizedmonomers so that the monomers surround the molecule to be imprinted. Inthe process, functionalized monomers align themselves in a bindingrelationship to complementary functional groups on the imprint molecule.The monomers are then polymerized, thereby encasing the imprint moleculewithin the polymer. The imprint molecule is then washed away, and theresulting material contains imprinted binding sites which are the“negative” of the imprint molecule. The complementary binding groups,arising from the functionalized polymer groups incorporated during theimprinting, are specifically positioned to enhance the preferentialsubstrate binding and, if desired, subsequent catalysis.

[0008] To date, the methods of molecular imprinting described above haveachieved only modest success in producing imprinted materials thatexhibit selectivity and catalytic activity. The reason for this is thatin order to be effective in wide scale use, antibody and enzyme mimicsmust have binding/active sites that are nearly homogeneous (inspecificity and activity), well formed (based on shape and reactivity),and easily accessed by reactant molecules (access is affected by shape,size and polarity of the channels leading to the catalytic site). Sitehomogeneity and site accessibility are both equally important. Theimprinted sites created by currently known methods are generally notvery accessible and are generally not homogeneous, that is, they oftenhave different binding affinities and/or reactivities. These problemsarise from the methods used in forming the polymer imprint and providingaccess to the binding sites. Using the conventional imprinting process,the imprinted sites are completely encased within the polymer. In orderto enable access to the sites, the polymer may be ground up, therebyexposing the sites. However, doing so causes the deformation of a largenumber of the binding sites and irreversibly alters theshape-specificity and the complementary binding of the sites, therebyadversely affecting their selectivity and activity. In an alternativemethod of enabling access to the imprinted sites, porogens (typicallyinert solvents) may be incorporated among the polymerizable monomers inthe imprinting process. After polymerization, the porogens are washedaway, creating pores that allow access to the binding sites. However, asthe porogens are removed, some of the structural integrity of thepolymer is lost, leading to deformation of the sites. The resultant lossin specificity and activity is similar to that observed as a result ofgrinding up the polymer.

[0009] Recently, efforts have been made to improve accessibility bycreating imprinted sites on silica or polymer surfaces. In general, thisapproach involves linking complementary hydrogen-bonding functionalizedsilanes to the imprint molecule and then creating the molecularrecognition site by attaching this “scaffolding” to the surface of asilica or polymer particle. After the imprint molecule is washed away, abinding site with affinity for specific molecules remains on the surfaceof the particle. This approach is described in the followingpublications incorporated herein by reference: Lele B. S, et al“Molecularly Imprinted Polymer Mimics of Chymotrypsin 1. Cooperative 9Effects and Substrate Specificity” React. Funct. Polym 39(1), 37-52(1999); Lele, B. S., “Molecularly Imprinted Polymer Mimics ofChymotrypsin 2. Functional Monomers and Hydrolytic Activity” React.Funct. Polym 40(3), 215-229 (1999); and Hwang K-O, et al,“Template-Assisted Assembly of Metal Binding Sites on a Silica Surface”,Mater. Sci. Eng. C, 3, 137 (1995).

[0010] This approach has some important limitations: First, thescaffolding process places the imprint molecule on the surface of theparticle. Consequently, this procedure imprints only the functionalityof the imprint molecule and not the molecule's shape. Additionally,there are limits to how much of the imprint molecule's functionality canbe imprinted using this procedure. This is essentially a 2-dimensionalform of imprinting in that only those functional groups of the imprintmolecule with pre-attached complementary binding groups oriented towardsthe particle surface would be imprinted. Functional groups withpre-attached complementary binding groups oriented away from the surfacewould not be tethered to the surface and so would not be imprinted. Thefewer functional groups imprinted, the lower the selectivity of theimprinted site for the target molecule, and the binding of the targetmolecule also will be much weaker. Second, because of the chemistryinvolved in attaching the imprint molecule-complementary groups complexto a surface, the “scaffolding” procedure is limited to the imprintingof particle or planar surfaces. This procedure is not useful forimprinting porous materials due to difficulties in introducing thereactants into the pores. Even if the attachment to the surfaces of thepores could be achieved, this imprinting would necessarily restrict theflow of any target molecules through the pores, thereby creating theaccessibility problems this approach was designed to alleviate.

[0011] In a separate field of technology, methods have been developedfor making particles and porous materials by template-directedsynthesis. In these methods, surfactants are used to create molecularmicrostructures such as micelles or reverse micelles in a solvent mediumand then inorganic or organic monomers are polymerized around themolecular microstructures at the surfactant-solvent interface. When thesurfactant is removed, the remaining material has a size and shapecomplementary to the size and shape of the molecular microstructures. Bycontrolling variables such as surfactant selection and concentration, avariety of different microstructure shapes such as micellar, cubic,tetragonal, lamellar, tubular and reverse micellar can be formed and,consequently, monodisperse particles of a variety of different sizes andporous materials with a variety of different shapes of pores andchannels can be created. Methods of making porous material aredescribed, for example, in the following patents and publicationsincorporated herein by reference: U.S. Pat. No. 5,250,282 to Kresge etal; U.S. Pat. No. 5,304,363 to Beck et al; U.S. Pat. No. 5,321,102 toLov et al; U.S. Pat. No. 5,538,710 to Guo et al; U.S. Pat. No. 5,622,684to Pennavaia et al; U.S. Pat. No. 5,750,085 to Yamada et al; U.S. Pat.No. 5,795,559 to Pinnavaia et al; U.S. Pat. No. 5,786,294 to Sachtler etal; and U.S. Pat. No. 5,858,457 to Brinker et al; J. C. Vartuli, et al,“Effect of Surfactant/Silica Molar ratios on the Formation of MesoporousMolecular Sieves: Inorganic Mimicry of Surfactant Liquid-Crystal Phasesand Mechanistic Implications” Chemistry of Materials, 6, 2317 2326(1994); C. A. Morris, et al “Silica Sol as a Nanoglue: FlexibleSynthesis of Composite Aerogels” Science, 284, 622-624 (1999); B. T.Holland et al, “Synthesis of Highly Ordered, Three-Dimensional,Macroporous Structures of Amorphous or Crystalline Inorganic Oxides,Phosphates and Hybrid Composites” Chem Mater 11, 795-805 (1999); and M.Antonietti, et al, “Synthesis of Mesoporous Silica with Large Pores andBimodal Pore Size Distribution by Templating of Polymer Latices”Advanced Materials 10, 154-159 (1998). These materials, while being ableto distinguish molecules on the basis of size, typically lack thespecificity and activity of enzymes and antibodies. Methods for makingmonodisperse silica particles by hydrolyzing alkoxysilanes in asurfactant-stabilized water-in-oil microemulsion containing ammonia aredescribed, for example, in the following patents and publicationsincorporated herein by reference: U.S. Pat. No. 5,209,998 to Kavassalisat al; W. Stober et al, “Controlled Growth of Monodisperse SilicaSpheres in the Micron Size Range” J. Colloid Interface Sci., 26, 62(1968); Lindberg et al, “Preparation of Silica Particles Utilizing theSol-Gel and the Emulsion-Gel Processes” Colloids and Surfaces A 99, 79(1995); P. Espiard et al, “A Novel Technique for Preparing OrganophilicSilica by Water-In Oil Microemulsions” Polymer Bulletin, vol. 24, 173(1990); H. Yamauchi et al, “Surface Characterization of UltramicroSpherical Particles of Silica Prepared by W/O Microemulsion Method”,Colloids and Surfaces, Vol 37, 71-80 (1989); Markowitz et al, “SurfaceAcidity and Basicity of Functionalized Silica Particles” Colloids andSurfaces A: Physicochem Eng. Aspects 150, 85-94 (1999). The formation ofsilica gel-coated metal and semiconductor nanoclusters is described inU.S. Pat. No. 5,814,370 to Martino et al. As described below,unfunctionalized silica particles have little or no catalytic activity;catalytic activity is increased with functionalized silica particles,but not to the level achieved with imprinted materials.

SUMMARY OF THE INVENTION

[0012] Accordingly, it is an object of the present invention to providean imprinted material and a method of making an imprinted materialwherein the imprinted sites are easily accessible to target molecules.

[0013] It is a further object of the present invention to provide animprinted material and a method of making an imprinted material whereinthe imprinted sites are nearly homogeneous in activity and specificity.

[0014] These and other objects are achieved by a method for making amolecularly imprinted solid structure by the steps of: providing asurfactant compound having a headgroup, wherein the headgroup comprisesa group to be molecularly imprinted; combining the surfactant compoundwith a solvent to form a mixture and maintaining the mixture from theprevious step so that molecules of the surfactant self-assemble to format least one supramolecular structure having exposed imprint groups;combining the mixture with at least one reactive monomer so that thesupramolecular structure serves as a template for the organizing ofmolecules of the reactive monomer surrounding or in contact with thesupramolecular structure, including surrounding the imprint groups;maintaining the mixture so that the reactive monomers react with eachother to form a solid structure having a shape complementary to theshape of the supramolecular structure, including the shape of theexposed imprint groups; and removing the supramolecular structure fromthe solid structure. The resulting structure is a porous material orparticle having molecularly imprinted sites. Because the imprinting isaccomplished into the material's surface, the imprinted sites are easilyaccessible to target molecules. And because no drastic processing stepssuch as grinding are required to achieve accessibility, the imprintedsites tend to be homogeneous in specificity and reactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the kinetics of the hydrolysis of DL-BAPNA catalyzedby non-imprinted silica particles () and silica particlessurface-imprinted with 10% (∘), 20% (▾), and 40% (Δ)N-α-decyl-L-phenylalanine-2-aminopyridine. All particles contain 5 wt %(total silica) of a mixture of PEDA, IPTS, and CTES.

[0016]FIG. 2 shows the kinetics of the hydrolysis of various substratesby silica particles surface-imprinted withN-α-decyl-L-phenylalanine-2-aminopyridine. All particles contain 5 wt %(total silica) of a mixture of PEDA, IPTS, and CTES.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention provides methods for making molecularlyimprinted particles and porous materials having accessible andhomogeneous imprinted sites.

[0018] As used herein, the term “molecularly imprinted” material refersto material that has been polymerized around molecules of an imprintcompound in such a way that when the imprint group is removed from thematerial, cavities or “imprinted sites” remain in the material that arenegative images of the imprint compound. When the molecularly imprintedmaterial is subsequently exposed to a solution containing the imprintcompound, the imprinted sites selectively bind the imprint compound.This selective binding allows the material to be used as an artificialantibody or enzyme. For even greater selectivity of the molecularlyimprinted material, functional monomers with complementary bindingaffinity to functional groups on the imprint compound can beincorporated into the material.

[0019] To make a molecularly imprinted material according to the presentinvention, an imprint group is coupled to, or forms the headgroup of, asurfactant. The surfactant is then maintained in a mixture underconditions in which the molecules of the surfactant form at least onesupramolecular structure and wherein the imprint groups are exposed onthe surface of the supramolecular structure. The solution is combinedwith reactive monomers (which can include functionalized monomers) undersufficient conditions so that the reactive monomers surround thesupramolecular structure, including the exposed imprint groups. Thereactive monomers are polymerized to form a solid structure that has thenegative shape of the supramolecular structure. That is, it has asurface topography that is complementary to the surface topography ofthe supramolecular structure. The supramolecular structure is thenremoved, and what is left behind is a solid structure that has pores,channels, or other types of voids corresponding to the shape of thesupramolecular structure. Because the imprint groups were exposed on thesurface of the supramolecular structure when the solid structure wasformed, the surfaces or walls of the voids include imprint sites, whichare indentations in the surface having a shape complementary to theshape of the imprint group. These imprint sites, because they arecreated on the surfaces or walls of voids, are extremely accessible,much more so than when the imprint group alone is used to create amolecularly imprinted material.

[0020] By this method, a porous structure having pores or channels andhaving molecularly imprinted indentations or cavities in the walls orsurfaces of the pores or channels may be created. Typically, the poresor channels may be in the size range of about 0.01 μm to about 0.5 μm orgreater

[0021] Alternatively, molecularly imprinted particles may be formed bycombining an imprint group-coupled surfactant as described above with anorganic solvent and water to form a water-in-oil microemulsion. In themicroemulsion, molecules of the surfactant arrange themselves so thatthe imprint groups are in the water phase and the hydrophobic tails arein the organic phase. When a water-soluble reactive monomer is added andcaused to polymerize in the water phase, the particles that are formedhave indentations in their surfaces that are the mirror image of theimprint groups. If functionalized reactive monomers are also added tothe microemulsion, the imprinted sites of the particles will includefunctional groups in a spaced-apart arrangement corresponding to thelocation of complementary functional groups on the imprint group. Bythis method, imprinted particles of a variety of sizes can be created.For example, imprinted particles having a narrow size distribution andhaving an average size of between about 50 and about 1200 nm can becreated.

[0022] The imprint group in the present invention can be any chemicalentity for which it would be useful to have imprinted sites. Forexample, if the imprinted material is to be used as an artificialantibody, the imprint group can be the compound or a portion of thecompound sought to be bound by the antibody. If the imprinted materialis to be used as an enzyme, the imprint group can be a transition stateanalog (TSA), that is, an analog of the transition state that is formedduring the enzymatic conversion of a substrate to a product. The imprintgroup may also be an inhibitor (a compound that inhibits enzymeactivity), a substrate or a substrate analog of the enzyme.

[0023] The surfactant in the present invention can be any compound suchas, for example, an amphiphilic compound, that is capable ofself-assembling in solution to form one or more supramolecularstructures or any monomeric or polymeric surfactant that can formlamellar and/or non-lamellar phases. The imprint group is coupled to thesurfactant by any sufficient means so that when molecules of thesurfactant form one or more supramolecular structures, the imprintgroups are exposed on the surface of the supramolecular structures.Preferably, the surfactant is an amphiphilic compound having ahydrophilic headgroup and a hydrophobic tail, and the imprint groupmakes up, or forms part of, the headgroup of the compound. (Such asurfactant can be created, for example, by acylating a hydrophilicimprint compound so that the combination of a hydrophilic headgroup anda hydrophobic acyl tail gives the compound the amphiphilic properties ofa surfactant.) Due to thermodynamic driving forces, molecules of anamphiphilic surfactant self-assemble in an aqueous medium to minimizethe exposure of their hydrophobic portions to the medium, whilemaximizing the exposure of the hydrophilic headgroup. Consequently, if asurfactant has an imprint group as a headgroup, in whole or in part, theimprint group will be exposed to the surfaces of the self-assembledstructure and imprinted sites will be formed when these surfaces aresubsequently mineralized or when a mirror-image polymer is created.

[0024] Alternatively, the imprint group could be attached to the end ofa hydrophobic portion of a surfactant. In this embodiment, a non-aqueoussolvent or a solvent combination having an oil phase would be selectedso that when a supramolecular structure is formed in a solvent, theimprint group would exposed be to the non-aqueous or oil phase. Theorganic or inorganic reactive monomers would be selected so that theypolymerize in a non-aqueous environment and imprinting would occur atthe surface of a polymer formed in the oil phase.

[0025] The imprint group-coupled surfactant is combined with a solventand the solution is maintained under conditions of temperature, time, pHand surfactant concentration so that molecules of the surfactantself-assemble into supramolecular structures. The conditions oftemperature, time, pH and surfactant concentration that are sufficientdepends on the particular surfactant and how it behaves in theparticular solvent. The sufficient conditions can be readily determinedby experimentation based on what is known about the behavior ofnon-coupled surfactants. Preferably, the surfactant is selected andpaired with a particular solvent wherein it is known that supramolecularstructures will form easily.

[0026] The solution may also contain at least one additional surfactant,that is, a surfactant that does not have an imprint group associatedwith it. The additional surfactant is selected so that it alsoself-assembles with the imprint group-coupled surfactant and becomesincorporated into the supramolecular structure. An additional surfactantwould be used, for example, in instances where the imprint group is arelatively-large molecule compared to the surfactant and wherein itwould be desirable to spread out the exposed imprint groups on thesurface of the supramolecular structure.

[0027] The term “supramolecular structure” includes any liquid crystalstructure formed by the arrangement of surfactant molecules in asolvent. Examples of structures include micelles, vesicles, bicontinuouscubic phase, hexagonal phase, tubules or reverse micelles. The size andtype of structures that are formed depends on variables such as thetemperature, pH, the concentration of the imprint group-surfactant, theidentity and concentration of any additional surfactant, the identity ofthe solvent, the identity and concentration of the reactive monomer,etc. Because the purpose of the supramolecular structure is to provideaccessibility for imprinted sites, it will often not be critical whichparticular liquid crystal structure is formed, as long as asupramolecular structure is formed that can serve as a template and thatcan hold the imprint groups in an exposed position on the surface of thesupramolecular structure. Likewise, in the formation of imprintedparticles in a water-in-oil microemulsion, the size of the particlesthat can be formed is affected by variables such as the temperature, pH,the concentration of the imprint group-surfactant, the identity andconcentration of any additional surfactant, the identity of the solvent,the identity and concentration of the reactive monomer, etc.

[0028] The mixture of the imprint group-coupled surfactant and solventis combined with reactive monomers. The reactive monomers may be addedeither before or after the supramolecular structures form. The mixtureis maintained under sufficient conditions of time, temperature andmonomer concentration so that the monomers surround the supramolecularstructure and so that the supramolecular structure acts as a template todirect the organizing and positioning of the monomers around thestructure. The sufficient conditions depend on the particular monomersused and are either well known for specific monomers or can be readilydetermined by experimentation. Because the imprint groups are exposed onthe surface of the supramolecular structure, the reactive monomerssurround the imprint groups as well. The addition of the inorganicmonomers may induce changes in the supramolecular structure from themicellar surfactant phase to a mesoporous surfactant phase (cubic orhexagonal) during the polymerization or mineralization process, but thiswould not affect the creation of imprinted sites. In this manner,mesoporous metal oxide materials can be formed in wter using an acid orbase catalyst. Cationic surfactants, such as cetyltrimethylammoniumbromide (CTAB), or nonionic surfactants, such as BRIJ® type (a class ofpolyoxyethylene alkyl ethers sold by ICI Americas Inc., Wilmingon, Del.)or amphiphilic block co-polymers, can be used to form theliquid-crystalline phases (LC) that transform to cubic or hexagonalphases upon complexation with the monomeric inorganic precursor.Subsequent hydrolysis and condensaton of this monomer, followed byremoval of the surfactant results in formation of the mesoporous metaloxide.

[0029] The reactive monomers can be molecules any compound or compoundsthat are capable of surrounding the supramolecular structure and thatcan be reacted with each other or polymerized to form a solid orshape-retaining structure with a shape that is complementary to theshape of the supramolecular structure and the exposed imprint groups.The monomers are selected according to the desired end product. Forexample, to make an inorganic oxide or metal oxide molecularly imprintedmaterial, the monomers may be inorganic oxide or metal oxide precursors.As a more specific example, to make a molecularly imprinted material ofsilica, the reactive monomers may be alkoxysilanes, particularlytetraalkoxysilanes such as tetraethoxysilane (TEOS), tetramethoxysilane(TMOS), tetrabutoxysilane (TBOS), etc. To make inorganic oxidescontaining germanium, titanium or aluminum, reactive monomers include,but are not limited to, tetramethoxygermane, tetraisoprpoxygermane,tetraethoxygermane, tetrabutoxygermane, aluminum n-butoxide, aluminumisoproxide, titanium ethoxide, titanium diisopropoxide(bis-2,4-pentanedionate), titanium methyl phenoxide, vanadiumtriisopropoxide oxide, vanadium tri-n-propoxide, zirconium n-butoxide,zirconium n-propoxide, etc. Mixed inorganic or metal oxides may be madeby combining inorganic precursors. To make an organic molecularlyimprinted material, the reactive monomers may be polymerizable organicmonomers including, but not limited to, acrylate, methyl methacrylate,olefins, pyrrole, saccharides, silanes, styrene, isocyanates, vinylacetate, vinyl chloride, etc. Mixed materials may be made by combininginorganic and organic reactive monomers.

[0030] The reactive monomers may include functionalized monomers, thatis, monomers that have functional moieties with complementary bindingaffinity for functional moieties on the imprint group by, for example,hydrogen-bonding, electrostratic interactions or other van der Waalsinteractions. Examples of functional moieties include, but are notlimited to, amine (primary, secondary, tertiary, quaternary), hydroxyl,carboxylate, sulfhydryl, amino acids, metal chelates such asiminodiacetic acid-metal ions (divalent or trivalent metal ions),ureido, urea, thiourea, amidine, methyl, phenyl, fluorocarbons,nucleotides, phosphonucleotides, phosphates, saccharides, multiplecombinations of any of the these (ie. dicarboxylates, diamines, etc),and multiple combinations of different functional groups. As thereactive monomers surround the supramolecular structure, thefunctionalized monomers will tend to become spatially distributed andoriented around the imprint groups so that they form binding complexeswith the corresponding functional moieties on the imprint group. Then,as the reactive monomers are polymerized, the functionalized monomersare locked in place. In the formation of a silica imprinted structureusing a tetraalkoxysilane such as tetraethoxysilane, examples offunctionalized monomers that can be used include3-(Aminoethylaminomethyl)-phenyltrimethoxysilane (PEDA),carboxyethylsilanetriol (CTES), andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole (IPTS),3-aminoalkyltriethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, decyltriethoxysilane,hydroxymethyltriethoxysilane, isobutylmethyldimethoxysilane,3-mercaptopropyltriethoxysilane,pentafluorophenylpropyltrimethoxysilane, phenyltrimethoxysilane,N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride,ureidopropyltriethoxysilane, and3-trihydroxysilyl-propylmethylphosphonate, sodium salt.

[0031] The sufficient conditions under which the monomers react variesdepending on the monomer and is either well known for a particularmonomer or can be readily determined by experimentation. Inorganicmonomers generally polymerize in water in the presence of a catalyst.For example, tetraethoxysilanes and similar monomers hydrolyze in waterat room temperature in the presence of an ethanol/ammonia catalyst.Organic monomers polymerize in the presence or absence of a catalyst atvarying temperatures, solvent mixtures, pressure, exposure to UVirradiation etc., depending on the particular monomer.

[0032] After the reactive monomers are reacted to form molecularlyimprinted particles or a molecularly imprinted porous structure, themolecules of the surfactant are extracted and removed. This can be doneby any means such as, for example, washing the particles or thestructure.

[0033] Having described the invention, the following examples are givento illustrate specific applications of the invention including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication

EXAMPLES

[0034] Molecular imprinting and template directed synthesis were used tocreate catalytic silica particles that have catalytic activity asesterase mimics. Specifically, L-phenylalanine-2-aminopyridine (achymotrypsin transition state analog (TSA)) and 3-aminophenyl-boronicacid (a chymotrypsin inhibitor) were acylated to form amphiphilicimprint molecules, which were then mixed with the nonionic surfactantpolyoxyethylene(5) nonylphenyl ether (NP-5). These surfactant mixtureswere used to form microemulsions that were then utilized to synthesizesurface imprinted silica particles from tetraethoxysilane (TEOS) andamine, carboxylate, and dihydroimidazole functionalized silanes. Theeffects of the surfactant TSA structure, surfactant TSA/NP-5 ratios, andfunctionalized silane/TEOS ratios on the catalytic activity of theparticles is described. In addition, the specificity of these particlesis discussed.

[0035] Materials and Equipment

[0036] 3-(Aminoethylaminomethyl)-phenyltrimethoxysilane (PEDA),carboxyethylsilanetriol (CTES), andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole (IPTS) were purchasedfrom Gelest (Tullytown, Pa.); Tetraethoxysilane (TEOS), Igepal® CO-520(polyoxyethylene(5) nonylphenyl ether) (NP-5), 3-aminophenylboronicacid, 2-aminopyridine, dicyclohexylcarbodiimide (DCC), trifluoroaceticacid (TFA), ethanol, cyclohexane, acetonitrile, and dimethyl sulfoxide(DMSO) were purchased from Aldrich Chemical Co. (Milwaukee, Wis.);N-α-t-BOC-L-phenylalanine was purchased from Calbiochem-NovabiochemCorp. (San Diego, Calif.); methanol and glacial acetic acid werepurchased from Fisher Scientific (Pittsburgh, Pa.); all chemicals wereused as received. Saturated solutions of ammonia in ethanol wereprepared by passing ammonia gas into denatured ethanol at 20° C. for 5-6hours. Electron microscopy was performed with a Zeiss EM-10 transmissionelectron microscope operated at 60 kV. UV/Vis spectroscopy was performedwith a Beckman DU-650 spectrophotometer.

[0037] Synthesis of Imprint Group-Coupled Surfactants

[0038] The following describes the synthesis of two compounds,N-α-decyl-L-phenylalanine-2-aminopyridine and 3-octylamidophenylboronicacid, that were used to create molecularly imprinted polymers bytemplate-directed synthesis. Each of these compounds is a surfactantthat comprises a headgroup and an acyl tail, the headgroup being theportion of the compound that is to serve as the imprint group.

[0039] Synthesis of N-α-decyl-L-phenylalanine-2-aminopyridine (asurfactant—α-chymotrypsin transition state analog). This compound hasthe following structure:

[0040] The acylated phenylalanine anilide imprint molecule wassynthesized in three steps. (1) N-α-t-BOC-L-phenylalanine was coupledwith 2-aminopyridine using DCC to giveN-α-t-BOC-L-phenylalanine-2-aminopyridine. (2) The α-amine was thendeprotected with TFA to give N-α-L-phenylalanine-2-aminopyridine.(3) Tocreate a compound having the properties of a surfactant, the free aminewas then acylated with decanoic acid using DCC to giveN-α-decyl-L-phenylalanine-2-aminopyridine.

[0041] (1) Synthesis of N-α-t-BOC-L-phenylalanine-2-aminopyridine: DCC(4.4 g, 21.1 mmol) was added to a solution of N-α-t-BOC-L-phenylalanine(10 g, 37.7 mmol) in 200 mL THF at 0° C. The mixture was stirred for 30minutes and then filtered. 2-Aminopyridine (4.5 g, 41.5 mmol) was addedto the filtrate in a round bottom flask and the mixture was stirredovernight. The reaction mixture was filtered and the solvent wasevaporated under reduced pressure until approximately 20 mL of THFremained. Petroleum ether was added and the product was precipitatedfrom solution overnight. The semi-solid precipitate collected byfiltration was purified by silica gel column chromatography (95:4:1CHCl₃:CH₃OH:H₂O, v/v/v) to give 8 g (60% yield) of the desired amide.

[0042] (2) Synthesis of N-α-L-phenylalanine-2-aminopyridine:-α-t-BOC-L-phenylalanine-2-amino-pyridine (2 g, 5.8 mmol) was dissolvedin 10 mL 1:1 TFA:CH₂Cl₂ (v/v) and stirred vigorously for 10 min. TheTFA:CH₂Cl₂ solution was then evaporated under a stream of N₂. Theresidue was dissolved in 1 mL CHCl₂ and purified by silica gel columnchromatography (80:18:2 CHCl₃:CH₃OH:H₂O, v/v/v) to give 1.4 g ofN-α-L-phenylalanine-2-aminopyridine in quantitative yield.

[0043] (3) Synthesis of N-α-decyl-L-phenylalanine-2-aminopyridine: DCC(1.0 g, 4.85 mmol) was added to a solution of decanoic acid (1.50 g, 8.7mmol) in 10 mL of chloroform under nitrogen atmosphere. A whitesuspension was formed after stirring for 3 hours at room temperature.The solid was removed by filtration. To the chloroform solution, 10 mLof a THF solution of N-α-L-phenylalanine-2-aminopyridine (1.0 g, 4.1mmol) was added. After stirring at room temperature for 12 hours, awhite suspension was formed. The solvents were removed under reducedpressure and the resulting solid was purified first by silica gel columnchromatography with a mixture of chloroform/methanol=95:5(v/v) as theeluent, then by crystallization in a mixture of hexanes and toluene togive N-α-decyl-L-phenylalamine-2-aminopyridine as a white crystallinesolid (11.1 g, yield: 67%).

[0044] Synthesis of 3-Octylamidophenylboronic acid (asurfactant—α-chymotrypsin inhibitor). This compound has the followingstructure:

[0045] Octanoyl chloride (4.6 g, 28.3 mmol) was added slowly to asolution of 3-aminophenylboronic acid (5.0 g, (26.9 mmol) in DMSO (50mL) containing pyridine (2.3 mL, 28.3 mmol). After addition wascomplete, the reaction mixture was stirred overnight. The reactionmixture was diluted with 500 mL water and extracted with chloroform. Theorganic fractions were collected and dried over MgSO₄, filtered, andthen, the solvent was evaporated under reduced pressure to give thecrude product as an oil. The oil was redissolved in a small amount ofchloroform and passed through a cation exchange column (Bio-Rad AG50W-X8resin, 20-50 mesh, hydrogen form) to remove any pyridine bound to theboronic acid. The crude product was then purified by silica gel columnchromatography (98:2 CHCl₃:CH₃OH, v/v) to give 2.5 g, (35.6% yield) ofthe desired product.

[0046] Synthesis of Particles

[0047] The following particles were synthesized:

[0048] Silica Particles Without Modification (Comparative Example)

[0049] Silica particles that did not contain functionalized silanes wereprepared by stirring a saturated solution of ammonia in ethanol withcyclohexane, the surfactant Igepal® CO-520 (polyoxyethylene(5)nonylphenyl ether) (NP-5), and water for 30 minutes at room temperatureand then adding tetraethoxysilane (TEOS). Stirring continued overnight.The volume of the reaction mixture was reduced by vacuum evaporation andthe particles were separated from the remaining reaction mixture bycentrifugation, washed three times with a wash solution consisting of 4parts methanol, one part glacial acetic acid, and one part water,followed by washing three times with acetonitrile. The particles werethen air dried overnight. Unstained, unwashed particles on copper gridswere observed by electron microscopy to determine particle size.

[0050] Silica Particles With Surface Amine, Dihydroimidazole, andCarboxylate Groups (Comparative Example)

[0051] Silica particles with surface amine, dihydroimidazole, andcarboxylate groups were prepared by including measured amounts of3-(aminoethylaminomethyl)-phenyltrimethoxysilane (PEDA),carboxyethylsilanetriol (CTES), andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole (IPTS) to the saturatedsolution of ammonia/ethanol, cyclohexane, Igepal® CO-520(polyoxyethylene(5) nonylphenyl ether) (NP-5), and water describedabove, before stirring and adding TEOS. The particles were separated anddried as described above.

[0052] Imprinted Silica Particles Made Without Functionalized Silanes

[0053] Imprinted silica particles were prepared by mixing NP-5 with animprint group-coupled surfactant and solubilizing in ethanol/cyclohexaneand then adding the mixture to the ammonia/ethanol mixture. TEOS wasthen added and particles were formed, separated and dried in the samemanner as described above.

[0054] Imprinted Silica Particles Made With Functionalized Silanes

[0055] Imprinted silica particles made with functionalized silanes wereprepared by mixing NP-5 with an imprint group-coupled surfactant andsolubilizing in ethanol/cyclohexane and then adding the mixture to theammonia/ethanol mixture.3-(Aminoethylaminomethyl)-phenyltrimethoxysilane (PEDA),carboxyethylsilanetriol (CTES), andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole (IPTS) were added beforestirring and TEOS was added. The particles were formed, separated anddried in the same manner as described above.

[0056] Assay for Hydrolytic Activity.

[0057] Hydrolytic activity of the silica particles was determined byobserving the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitrophenyl ester(Suc-AAPF-PNP), a chymotrypsin substrate, andbenzoyl-DL-arginine-p-nitrophenyl ester (DL-BAPNA), a standardchromogenic substrate of trypsin.

[0058] Suc-AAPF-PNP has the following structure:

[0059] L-BAPNA has the following structure:

[0060] D-BAPNA has the following structure:

[0061] Substrate stock solutions were prepared by dissolving ˜50 mgBAPNA or suc-AAPF-PNP in DMSO; these stock solutions were then dilutedwith DMSO and 0.1 M TRIS/HCl buffer (pH 7.4) to provide substratesolutions of various concentrations (final concentration of DMSO in eachsubstrate solution was 15% v/v). A measured quantity of particles;30-100 mg, were placed in a microcentrifuge tube, 1.3 ml of substratesolution was added, and the mixture was bath-sonicated to completelydisperse the particles. The mixture was then placed in a water bath at30° C. for 5-6 hours, with additional agitation provided every 45-60minutes. The mixture was then removed from the water bath andcentrifuged at 14,000 rpm for 30 minutes. The reaction time was measuredfrom initial sonication to the beginning of centrifugation. Catalyticactivity was determined from the increase in free p-nitrophenolconcentration, measured by UV/VIS spectroscopy at 410 nm with anextinction coefficient of 8500 M⁻¹ cm⁻¹. The measured absorbance wascompared to that of the substrate solution at 410 nm and from theabsorbance due to light scattering of the particle dispersion ofparticles in 0.1 M Tris buffer (prepared identically as above) at 410nm. These contributions to the absorbance were subtracted from the totalabsorbance at 410 nm to obtain the absorbance due to free p-nitrophenol.Each data point was calculated based on the average of 3 to 7 trials.

[0062] Differentiation of Catalytic Activity After Imprinting inNon-Functionalized and Functionalized Silica Particles

[0063] Using the above procedure, the catalytic activity offunctionalized and non functionalized silica particles imprinted withN-α-decyl-L-phenylalanine-2-aminopyridine was measured. The substratewas Suc-AAPF-PNP at a concentration of 0.2 mM. The reactions wereperformed in Tris buffer (50 mM), pH 7.8. in a volume of 1.0 ml. Rateswere converted from Absorbance (410 nm) units into 11 mol units via astandard curve with p-nitroanilide. The data were normalized per mg ofcatalyst.

[0064] Rates are given as μmol nitroanilide/(mg catalyst min). The rateof hydrolysis using imprinted particles containing 5% amine silane, 5%carboxylate silane, and 5% imidazole silane was 5.2×10⁻⁵; the rate ofhydrolysis using imprinted particles with no amine, carboxylate, orimidazole groups was 1.0×10⁻⁵; the rate of hydrolysis using nonimprinted particles containing 5% amine silane, 5% carboxylate silane,and 5% imidazole silane was 3.0×10⁶; the rate of hydrolysis using nonimprinted particles without any functional groups was 1.9×10⁻⁶. A tableof these results is given below: TABLE 1 Initial rate of hydrolysis ofSuc-AAPF-PNP catalyzed by functionalized and non-functionalizedimprinted silica particles Description Initial Rate^(a) Ratio^(b) 5%PEDA + 5% CTES + 5% IPTS + 20% TSA 5.2 × 10⁻⁵ 27.3 No modifiers + 40%TSA 3.4 × 10⁻⁵ 17.9 No modifiers + 20% TSA 1.0 × 10⁻⁵ 5.3 5% PEDA + 5%CTES + 5% IPTS, No TSA 3.0 × 10⁻⁶ 1.6 No modifiers and no TSA 1.9 × 10⁻⁶1.0

[0065] This example demonstrates that catalytic activity is greater forimprinted particles with functional groups and that non imprintedparticles have far less catalytic activity.

[0066] Differentiation of Catalytic Activity of Silica ParticlesImprinted With Different Enzyme Transition State Analogs:

[0067] Using the above procedure, the catalytic activity offunctionalized and non functionalized silica particles imprinted withN-α-decyl-L-phenylalanine-2-aminopyridine 3-octylamidophenylboronic acidand BOC-L-Phe-2-aminopyridine (a non-acylated imprint molecule used as acomparative example), was measured. The substrate was DL-BAPNA, asolution of which was prepared by dissolving 400-500 mg BAPNA in 5 mldimethyl sulfoxide, and diluting 0.1 ml aliquots of this preparation to10 ml with 0.1 M Tris buffer (pH 7.4) to yield a ˜0.2 mM BAPNA solution.The trypsin catalyzed rate of substrate BAPNA hydrolysis has beencalculated from a literature report as 6.7 μM mg⁻¹ min⁻¹ at 25° C. Esterhydrolysis was performed at 30° C. Three to seven hydrolysismeasurements were made for each type of particle, and an analysis ofvariance at a 95% confidence level indicates that improvement inhydrolysis rate is provided by both the presence of the functionalsilanes on the particle surface and the presence of the imprint moleculeduring particle synthesis, and that there is a significant correlationbetween these two factors. A table of the initial rate data is givenbelow. TABLE 2 Initial rate data for the hydrolysis of DL-BAPNAcatalyzed by silica particles surface- imprinted with different imprintmolecules. Wt. % of Added Initial Rate α Functionalized (μM/mg/ (μM/mg/Imprint Molecule Silanes^(a) min*10⁵) min*10)^(b) None 0 0.47 0.128 None5 0.83 0.302 3-Octylamidophenyboronic 0 1.04 0.26 acid3-Octylamidophenyboronic 5 1.81 0.324 acid BOC-L-Phe-2- 0 1.30 0.162aminopyridine BOC-L-Phe-2- 5 1.41 0.156 aminopyridine N-Decyl-L-Phe-2- 01.37 0.302 aminopyridine N-Decyl-L-Phe-2- 5 3.98 0.489 aminopyridine

[0068] Particles surface-imprinted with the acylated phenylalanine andaminophenylboronic acid imprint molecules demonstrate an increasedenhancement of hydrolytic activity in the presence of functional surfacesilanes. In contrast, particles surface-imprinted with non-acylatedphenylalanine in the presence of functionalized silanes do not reveal asimilar rate enhancement.

[0069] Effect of Increasing Amount of Functional Silanes and ImprintMolecule on Hydrolytic Activity.

[0070] Using the above procedure, the catalytic activity offunctionalized silica particles imprinted with increasing amounts ofN-α-decyl-L-phenylalanine-2-aminopyridine was measured. The followingtable summarizes how the initial rate of hydrolysis of DL-BAPNA isaffected by differences in the amount of functional surface silanes andamount of imprint molecule used during the preparation of catalyticparticles: TABLE 3 Initial rate data for the hydrolysis of DL-BAPNAcatalyzed by silica particles surface-imprinted withN-α-decyl-L-phenylalanine-2-aminopyridine. Amount of Amount of ImprintMol- Functionalized Initial ecule (Mol % Silanes^(a) Substrate Rate α ofTotal (Wt % of Concentration (μM/mg/ (μM/mg/ Surfactant) Total Silica)(mM) min*10⁵ min*10⁵ 0 5 0.2 0.83 0.30 10 5 0.2 2.65 0.42 10 5 0.4 3.410.33 10 10 0.4 3.92 0.89 10 15 0.4 4.17 0.38 20 5 0.2 3.98 0.49 20 5 0.45.55 0.15 20 10 0.4 4.12 0.37 40 5 0.2 7.11 0.97 40 5 0.4 9.13 0.32 4015 0.4 6.28 0.62

[0071] With three to seven hydrolysis measurements for each particletype, an analysis of variance on the results obtained in 0.4 mMsubstrate solution indicates that there is no significant difference inhydrolysis rate resulting from increasing the amount of functionalsilanes present in the particle; therefore, although the presence of asmall amount of functional silanes in the catalytic particle providessome improvement in hydrolysis rate, there is no added benefit fromincreasing the amount of functional silanes beyond a certain level.There is, however, a significant benefit from increasing the amount ofimprint molecule used in the synthesis of the catalytic particles. Itwas found that this effect is linear up to 40% added imprint molecule inthe NP-5 surfactant used in the microemulsion to prepare the particles.

[0072] Evaluation of Kinetic Parameters for Catalytic Particles.

[0073] The catalytic activity of imprinted silica particles was studiedover a range of initial substrate concentrations, from about 0.1 mM to1.0 mM. FIG. 1 shows data for a series of silica particles synthesizedwith 5% incorporated functional silanes (PEDA, IPTS, CTES mixture) and0, 10, 20, and 40% added acylated phenylalanine-2-aminopyridine anilideimprint molecule. Data for hydrolysis catalyzed by non-imprinted silicais included in the plot for comparison although this data is notexpected to follow saturation kinetics. Solid lines in the figurerepresent curves fitting the data to the Michaelis-Menten equation,$v = \frac{K_{1}S}{K_{2} + S}$

[0074] using parameters for K₁ and K₂ derived from non-linearregression. The following table summarizes K₁ and K_(m) parameters for anumber of different imprinted silicas: TABLE 4 Kinetic constants for thehydrolysis of DL-BAPNA catalyzed by non-imprinted silica particles andby silica particles surface-imprinted withN-α-decyl-L-phenylalanine-2-aminopyridine. Amount of Amount of ImprintFunctionalized Molecule Silanes (Mol % of (Wt % of K₁ α Total Total(μM/mg/ (μM/mg/ K₂ α K₁/K₂ Surfactant) Silica) min*10⁵) min*10⁵) (mM)(mM) (mg⁻¹min⁻¹) 10 5  7.29 0.71 0.48 0.10 0.15 20 0  5.24 0.78 0.650.18 0.08 20 5  9.41 0.05 0.34 0.04 0.28 40 5 12.5 0.08 0.17 0.03 0.74

[0075] One observed trend is that the value of K₁/K₂, a measure ofcatalytic efficiency per mg of silica, increases with increasing amountof imprint molecule used during particle synthesis, as well as whenfunctional silanes are present in the catalyst.

[0076] Selectivity of Molecularly Imprinted Catalysts to SeveralSubstrates.

[0077]FIG. 2 presents data for the hydrolysis of racemic trypsinsubstrate DL-BAPNA, the optically pure substrates D- and L-BAPNA, andthe α-chymotrypsin substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.Solid lines in the FIG. 2 represent curves fitting the data to theMichaelis-Menten equation. The following table presents K₁, K₂ and K₁/K₂for succinyl-Ala-Ala-Pro-Phe-p-nitrophenol and D- and DL-BAPNA: TABLE 5Selectivity of catalysis by silica particles surface-imprinted withN-α-decyl-L-phenylalanine-2-aminopyridine. K₁ α K₂ α K₁/K₂ Substrate(μM/mg/min*10⁵) (μM/mg/min*10⁵) (mM) (mM) (mg⁻¹min−1) Suc-AAPF-NA 2.700.69 0.52 0.24 0.05 DL-BAPNA 9.41 0.48 0.34 0.24 0.28 D-BAPNA 9.43 0.540.24 0.24 0.39 L-BAPNA 0.65 0.11 0.55 0.21 0.01

[0078] There are two interesting features of this data set. First, thecatalyst particle seems to be selective for the trypsin substrateD-BAPNA over the chymotrypsin substrate, even though the imprintmolecule more closely mimics the chymotrypsin substrate. Second, theparticles appear to be selective for the D isomer of the trypsinsubstrate, even though the imprint molecule had the L-isomerconfiguration. In fact, the L-BAPNA is almost completely inert to theparticles.

[0079] These results demonstrate that template-directed molecularimprinting is a viable method of creating robust, enantioselectivecatalytic silica particles. To our knowledge, this is the first exampleof forming catalytic silica particles by imprinting reactive sitesexclusively into the surface of the particle. Our efforts represent analternative to conventional molecular imprinting techniques.Surface-imprinting was clearly demonstrated by the effect of increasingthe amount of acylated imprint molecule,N-decyl-L-phenylalanine-2-aminopyridine, on initial rate and K₁/K₂(measure of catalytic efficiency per amount of surface-imprinted silica)values of DL-BAPNA amide hydrolysis (Tables 2 and 3). As the amount ofN-decyl-L-phenylalanine-2-aminopyridine used to surface-imprint thesilica particles increased, the catalytic efficiency of the particlesincreased. For this to happen, the imprint molecule must act as asurfactant headgroup that is positioned at the surfactant-waterinterface of the microemulsion to create the catalytic site. Inaddition, the observation of enantioselectivity (Table 4) stronglysuggests that defined, surface-imprinted catalytic sites have beenformed using template-directed molecular imprinting.

[0080] While both the imprint molecule and the functionalized amine andcarboxylate silanes have a positive effect on the hydrolysis ratesobtained, it is their combination that produces the best catalyticparticles. The importance of the functionalized silanes can be seen fromthe data in Tables 1 and 2. The initial rates for particles imprinted inthe absence of functionalized silanes indicate that imprint moleculeshape has only limited impact on the efficacy of the molecularrecognition site created. Incorporation of functionalized silanes intothe particles reveals two important features of this imprintingmethod: 1) The lack of a rate enhancement for the non-acylatedchymotrypsin TSA in the presence of silanes as compared to the rateenhancement observed for the actuated imprint molecules stronglysuggests that acylation aids in effectively positioning the imprintmolecule at the surfactant-water interface at which the particle forms;2) the presence of complementary hydrogen-bonding silanes at thesurfactant-water interface as the silica particle is forming isessential to forming reactive catalytic imprints. The results suggestthat some or all of the surface amine and carboxylate groups, along withthe surface hydroxyl groups of the silica particles, are capable ofinteracting with the substrate amide carbonyl group to enhance itsreactivity. The observation that shape of the imprinted cavity alonedoes not produce effective imprinted catalytic sites within polymers hasbeen previously reported.

[0081] Results obtained to determine the effect of increasing the wt. %of the functionalized silanes in the imprinted silica particles revealthat doing so results in a decrease in rate of amide hydrolysis (Table2). This is probably due to hydrogen bonding between the functionalizedsilanes leaving fewer, available for interaction with the substrate.From previous reports, it is known that surface functional groups willform hydrogen bonds with the native surface hydroxyl groups of thesilica particles as well as with each other, thereby decreasing theirbasicity and ability to hydrogen bond to other molecules.

[0082] The most interesting kinetic data deal with the specificityobserved for amide hydrolysis catalyzed by silica particles withsurfaces imprinted with N-decyl-L-phenylalanine-2-aminopyridine. Thesurface imprinted particles have selectivity for the trypsin substrateover the chymotrypsin substrate even though imprinting was done with thechymotrypsin TSA (Table 4, FIG. 4). The magnitude of the kineticconstants obtained for the hydrolysis of L-, D-, and DL-BAPNA catalyzedby the surface-imprinted silica particles are consistent with aD-enantioselective mode of hydrolysis. In fact, the hydrolysis ofD-BAPNA catalyzed by the surface-imprinted particles is 10 times faster(K₁) and 39 times more efficient (K₁/K₂) than the hydrolysis of L-BAPNAcatalyzed by the particles. Based on the observed enantioselectivity, atleast three of the groups surrounding the chiral methine carbon ofD-BAPNA must be bound to the catalytic site surface-imprinted silicaparticle. This reversal of stereoselectivity has been observed to occurfor substrate hydrolysis or transformation catalyzed by enzymes such as-chymotrypsin, lipase, peptidases and -lactamases. Theenantioselectivity of the hydrolysis strongly suggests that molecularstructure affected substrate packing within the catalytic site.Substrate substituent effects have been observed to have a major factorinfluencing enzyme enantioselectivity. Since the substrate isstructurally different than the imprinting molecule, the observedenantiopreference of the amide hydrolysis catalyzed by thesurface-imprinted silica particles may arise because the D-BAPNA packsmore readily into the imprinted catalytic site than L-BAPNA.

[0083] Obviously, many modifications and variations of the presentinvention are possible in-light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically described.

What is claimed is:
 1. A method for making a material having amolecularly imprinted surface, the method comprising the steps of (a)providing a surfactant coupled to an imprint group, (b) combining thesurfactant with a solvent to form a mixture and maintaining the mixtureso that molecules of the surfactant self-assemble to form at least onesupramolecular structure having a surface and wherein the imprint groupsare exposed on the surface, (c) combining the mixture from step (b) withat least one reactive monomer so that the supramolecular structureserves as a template for the organizing of molecules of the reactivemonomer along the surface of the supramolecular structure and around theexposed imprint groups, (d) maintaining the mixture from step (c) sothat the reactive monomers react with each other to form a solidstructure having a surface topography complementary to the surfacetopography of the supramolecular structure and having indentationscomplementary to the shape of the exposed imprint groups, and (e)removing the supramolecular structure from the solid structure.
 2. Amethod for making a material having a molecularly imprinted surface, themethod comprising the steps of (a) providing a surfactant compoundhaving a headgroup portion, wherein the headgroup portion comprises animprint group, (b) combining the surfactant compound, a solvent and atleast one reactive monomer to form a mixture and maintaining the mixtureso that molecules of the surfactant compound self-assemble to form atleast one supramolecular structure having a surface wherein the imprintgroups are exposed on the surface and so that the supramolecularstructure serves as a template wherein the molecules of the reactivemonomer self-organize along the surface of the supramolecular structureand around the exposed imprint groups, (c) maintaining the mixture fromstep (b) so that the reactive monomers react with each other to form asolid structure having a surface topography complementary to the surfacetopography of the supramolecular structure having indentationscomplementary to the shape of the exposed imprint groups, and (d)removing the supramolecular structure from the solid structure.
 3. Themethod of claim 2 wherein the surfactant compound is a compound that ismade by the steps of providing a hydrophilic imprint compound andacylating the hydrophilic imprint compound to create an amphiphilicsurfactant compound.
 4. The method of claim 2 wherein the supramolecularstructure is in the form of a micelle, reverse micelle, vesicle,bicontinuous cubic phase structure, hexagonal phase structure, ortubule.
 5. The method of claim 2 wherein the reactive monomer is anorganic polymer precursor.
 6. The method of claim 2 wherein the reactivemonomer is selected from the group consisting of acrylate, methylmethacrylate, olefins, pyrrole, saccharides, silanes, styrene,isocyanates, vinyl acetate, vinyl chloride and combinations thereof. 7.The method of claim 2 wherein the reactive monomer is an inorganic oxideprecursor.
 8. The method of claim 2 wherein the reactive monomer isselected from the group consisting of tetraethoxysilane,tetramethoxysilane, tetrabutoxysilane, tetramethoxygermane,tetraisoprpoxygermane, tetraethoxygermane, tetrabutoxygermane, aluminumn-butoxide, aluminum isoproxide, titanium ethoxide, titaniumdiisopropoxide (bis-2,4-pentanedionate), titanium methyl phenoxide,vanadium triisopropoxide oxide, vanadium tri-n-propoxide, zirconiumn-butoxide, zirconium n-propoxide and combinations thereof.
 9. Themethod of claim 2 wherein the reactive monomer is a silicon oxideprecursor.
 10. The method of claim 2 wherein the reactive monomer is analkoxysilane. 11 The method of claim 2 wherein the mixture of step (b)includes a non-functionalized reactive monomer, a reactive monomerhaving an amine functional group, a reactive monomer having a carboxylfunctional group and reactive monomer having a dihydroimidazolefunctional group.
 12. The method of claim 2 wherein the mixture of step(b) includes a non-functionalized reactive monomer and at least onereactive monomer having a functional moiety selected from groupconsisting of primary amines, secondary amines, tertiary amines,quaternary amines, hydroxyl, carboxylate, sulfhydryl, amino acids, metalchelates, ureido, urea, thiourea, amidine, methyl, phenyl,fluorocarbons, nucleotides, phosphonucleotides, phosphates, saccharidesand combinations thereof.
 13. The method of claim 2 wherein the mixtureof step (b) includes at least one additional surfactant.
 14. The methodof claim 2 wherein, in step (b), the surfactant and the solvent arecombined first and the mixture of the surfactant and solvent ismaintained so that molecules of the surfactant compound self-assemble toform at least one supramolecular structure having exposed imprint groupsand wherein subsequently, the reactive monomer is added to the mixture.15. The method of claim 2 wherein the imprint group includes at leastone functional moiety, wherein the mixture of claim (b) includes atleast one reactive monomer that has a complementary functional moietythat binds reversibly to at least one functional moiety of the imprintgroup, wherein, in step (c), as molecules of the reactive monomersself-organize along the surface of the supramolecular structure andaround the exposed imprint groups, the molecules of the reactivemonomers having complementary functional moieties become spatiallydistributed with respect to molecules of the imprint group so that thecomplementary functional moieties of the reactive monomers bindreversibly to the functional moieties of molecules of the imprint group,and wherein, in the solid structure formed in step (d), the spatialdistribution of the reactive monomers having complementary functionalmoieties is retained.
 16. A molecularly imprinted solid structure madeby a method comprising the steps of (a) providing a surfactant coupledto an imprint group, (b) combining the surfactant with a solvent to forma mixture and maintaining the mixture so that molecules of thesurfactant self-assemble to form at least one supramolecular structurehaving a surface and wherein the imprint groups are exposed on thesurface, (c) combining the mixture from step (b) with at least onereactive monomer so that the supramolecular structure serves as atemplate for the organizing of molecules of the reactive monomer alongthe surface of the supramolecular structure and around the exposedimprint groups, (d) maintaining the mixture from step (c) so that thereactive monomers react with each other to form a solid structure havinga surface topography complementary to the surface topography of thesupramolecular structure and having indentations complementary to theshape of the exposed imprint groups, and (e) removing the supramolecularstructure from the solid structure.
 17. A molecularly imprinted solidstructure made by a method comprising the steps of (a) providing asurfactant compound having a headgroup portion, wherein the headgroupportion comprises an imprint group, (b) combining the surfactantcompound, a solvent and at least one reactive monomer to form a mixtureand maintaining the mixture so that molecules of the surfactant compoundself-assemble to form at least one supramolecular structure having asurface wherein the imprint groups are exposed on the surface and sothat the supramolecular structure serves as a template wherein themolecules of the reactive monomer self-organize along the surface of thesupramolecular structure and around the exposed imprint groups, (c)maintaining the mixture from step (b) so that the reactive monomersreact with each other to form a solid structure having a surfacetopography complementary to the surface topography of the supramolecularstructure having indentations complementary to the shape of the exposedimprint groups, and (d) removing the supramolecular structure from thesolid structure.
 18. A molecularly imprinted material comprising ainorganic oxide structure having a plurality of pores or channels,wherein the pores or channels have walls that define a plurality ofmolecularly imprinted cavities.
 19. The molecularly imprinted materialof claim 18 wherein the inorganic oxide structure is silica.
 20. Themolecularly imprinted material of claim 18 wherein the pores or channelsare in the size range of about 0.01 μm to about 0.5 μm.
 21. Themolecularly imprinted material of claim 18 wherein the walls that definea plurality of molecularly imprinted cavities include a plurality offunctional groups in a spaced-about location on the walls of themolecularly imprinted cavities, the spatial location of the functionalgroups being complementary to the spatial location of complementaryfunctional groups of a predetermined imprint molecule.
 22. A compositioncomprising inorganic oxide particles wherein the surface of eachparticle defines a plurality of molecularly imprinted cavities.
 23. Thecomposition of claim 22 wherein the inorganic oxide particles aresilica.
 24. The composition of claim 22 wherein the inorganic oxideparticles have an average size of between 50 nm and 1200 nm.
 25. Thecomposition of claim 22 wherein the surface of each inorganic oxideparticle includes a plurality of functional groups in a spaced-aboutlocation on the surface of the molecularly imprinted cavities, thespatial location of the functional groups being complementary to thespatial location of complementary functional groups of a predeterminedimprint molecule.
 26. A method of making molecularly imprinted particlescomprising the steps of (a) providing a surfactant compound having ahydrophilic headgroup portion and a hydrophobic tail portion, whereinthe headgroup portion comprises an imprint group, (b) combining thesurfactant compound with (i) an organic solvent immiscible with waterand capable of forming a stable microemulsion with water, and (ii) waterto form a microemulsion of water domains within a continuous phase ofthe organic solvent, wherein molecules of the surfactant occupy theboundaries between the water domains and the organic solvent and whereinthe molecules of the surfactant are oriented so that the imprint groupsextend into the water domains, (c) adding at least one reactive monomerto the microemulsion wherein the reactive monomer polymerizes to form asolid particle in each water domain, each solid particle beingsurrounded by molecules of the surfactant and the surface of the solidparticle having cavities complementary to the shape of the imprintgroup, and (d) isolating the solid particles and removing thesurfactant.
 27. A method of making molecularly imprinted silicaparticles comprising the steps of (a) providing a surfactant compoundhaving a hydrophilic headgroup portion and a hydrophobic tail portion,wherein the headgroup portion comprises an imprint group, (b) combiningthe surfactant compound with an organic solvent immiscible with waterand capable of forming a stable microemulsion with water, a hydrolyzingreagent, and water to form a microemulsion of water domains within acontinuous phase of the organic solvent, wherein molecules of thesurfactant occupy the boundaries between the water domains and theorganic solvent and wherein the molecules of the surfactant are orientedso that the imprint groups extend into the water domains, (c) adding atleast one alkoxysilane compound to the microemulsion wherein thealkoxysilane reacts with the hydrolyzing reagent to form a silicaparticle in each water domain, each silica particle being surrounded bymolecules of the surfactant and the surface of the silica particlehaving cavities complementary to the shape of the imprint group, and (d)isolating the silica particles and removing the surfactant.
 28. Themethod of claim 27 wherein the microemulsion of step (b) includes atleast one additional surfactant.
 29. The method of claim 27 wherein themicroemulsion of step (c) includes at least one non-functionalizedalkoxysilane and at least one functionalized alkoxysilane.
 30. Themethod of claim 27 wherein the microemulsions of step (c) includes atleast one non-functionalized alkoxysilane and at least one alkoxysilanehaving a functional moiety selected from group consisting of primaryamines, secondary amines, tertiary amines, quaternary amines, hydroxyl,carboxylate, sulfhydryl, amino acids, metal chelates, ureido, urea,thiourea, amidine, methyl, phenyl, fluorocarbons, nucleotides,phosphonucleotides, phosphates, saccharides and combinations thereof.31. The method of claim 27 wherein the microemulsion of step (c)includes a non-functionalized alkoxysilane, an alkoxysilane having anamine functional group, an alkoxysilane having a carboxyl functionalgroup and an alkoxysilane having a dihydroimidazole functional group.32. The method of claim 27 wherein the microemulsion of step (c)includes tetraethoxysilane and at least one functionalized alkoxysilaneselected from the group consisting of3-(aminoethylaminomethyl)-phenyltrimethoxysilane,carboxyethylsilanetriol, andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole,3-aminoalkyl-triethoxysilane,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, decyltriethoxysilane,hydroxymethyl-triethoxysilane, isobutylmethyldimethoxysilane,3-mercaptopropyltriethoxysilane,pentafluorophenylpropyltrimethoxysilane, phenyltrimethoxysilane,N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride,ureidopropyltriethoxysilane, and3-trihydroxysilyl-propylmethylphosphonate, sodium salt.
 33. The methodof claim 27 wherein the microemulsion of step (c) includestetraethoxysilane, 3-(aminoethylaminomethyl)-phenyltrimethoxysilane,carboxyethylsilanetriol, andN-(3-triethoxysilylpropyl)-4,5-dihydroimidazole.